The nucleotide sequence of the human genome will provide health benefits ranging from new diagnostic tools to therapeutic reagents. One of the most widely used analytical methods in genomic analysis is DNA sizing, primarily based on gel electrophoresis. The broad long-term goal of the present proposal is to develop extremely sensitive, rapid and high throughput DNA sizing methods based on atomic force microscopy (AFM). The AFM is at present the only practical single molecule technology that can accurately determine the lengths of DNAs that are several kilobases or smaller. It is also the only technology with the signal-to-noise to detect single DNA molecules, or single proteins bound to DNA, without contrast enhancing agents. This allows for a reduction in sample size of several orders of magnitude, compared with gel based methods. In addition, an immobilized DNA sample can be sized in less than 2 minutes with current instrumentation. Thus the AFM offers several potential advantages over present and competing technologies. The investigators propose to develop a simple high-density sample deposition system for AFM-based DNA sizing, and implement the use of an automated AFM for imaging arrays of target DNAs adsorbed to a surface. DNA samples requiring sizing will be immobilized in dense arrays on a solid support and imaged by AFM. The automated imaging will utilize a high-precision sample stage that can position an AFM tip to within 1 micrometer over a 14-inch distance. Pattern recognition software will determine the distribution of DNA lengths, and produce a size histogram similar to densitometric scans of gels. To further exploit the capabilities of the AFM, the investigators will develop approaches to decorate DNA fragments in sequence-specific patterns using proteins, nucleic acids or other sequence-specific reagents, that will specify the identity of a DNA fragment with some level of confidence. Conventional restriction mapping reveals a sequence dependent distribution of restriction sites, and is an indirect form of DNA decoration. The ability to directly visualize stalled restriction enzymes by AFM can be used to produce decoration patterns that directly correspond to restriction maps, and other decoration strategies will provide unique coding patterns. Pattern recognition software will be developed to determine decoration patterns, and software that compares patterns will also be developed. The combination of DNA decoration and solid state sizing will be used to order sets of plasmids derived from BACs or PACs. Such ordered sets will reduce the amount of redundant sequencing, and facilitate the filling of gaps generated during shotgun sequencing.